Despite the effectiveness of antiretroviral therapy, HIV-associated neurocognitive disorders (HAND) that affect HIV infected individuals continue to increase. The prevalence of HAND and the incomplete reversal of neurocognitive dysfunctions after antiretroviral therapy have called for novel therapeutic approaches. Among the various pathophysiology of HAND, synaptic dysfunction likely underlies cognitive impairments. Interestingly, Tat, an essential HIV-1 viral protein, is present in the cerebrospinal fluid of individuals virologically controlled on cART. Furthermore, Brain-specific HIV protein Tat expression in mice mimics key aspects of HAND pathology in the post-cART era, suggesting that Tat may be responsible for the sustained central nervous system complications in patients receiving cART. Tat is known to cause neuronal injury via excitotoxic mechanisms. Furthermore, HIV-1-infected patients have significantly higher concentrations of glutamate in their plasma and cerebrospinal fluid compared to uninfected controls. Elevated levels of glutamate disrupt normal neural transmission in the brain, contributing to the neuropathogenesis of HIV-1 infection. In the past decade we have established that blocking the activity of glutaminase (GLS), a primary enzyme for the production of glutamate, could alleviate macrophages and microglia neuroinflammatory and neurotoxic response. We have demonstrated causal effects of innate immune activation and proinflammatory on the GLS function in macrophages, microglia, and neurons. Furthermore, we have observed an intriguing release of GLS by macrophages, microglia, and neurons, through unidentified mechanism(s) that could cause neuronal injury. Extracellular vesicles (EVs), which include microvesicles and exosomes, have emerged as an important cellular mechanism for GLS release. Therefore, in the current proposal, we hypothesize that the release of GLS-containing EVs is a critical pathogenic event in HIV-1-mediated neuronal injury and hippocampal synaptic dysfunction. Moreover, we hypothesize that blocking aberrantly upregulated/released GLS through GLS inhibitors could have therapeutic effects in HAND. Information will be provided as whether brain-specific overexpression of GLS is sufficient to induce brain inflammation, impair synaptic integrity and cognition in mice, and whether macrophage-specific conditional knockout of GLS gene and blocking of GLS-containing microvesicles release could protect neuronal function in a Tat transgenic mouse model of HAND. Furthermore, novel water-soluble GLS inhibitors will be evaluated for their therapeutic potentials in HAND relevant animal models. The elucidation of the GLS dysregulation and its contribution to pathophysiology of HAND will aid in developing potential novel agents for the treatment of HAND and other neurodegenerative disorders.